Yiqi Huoxue Decoction attenuates ischemia/hypoxia-induced oxidative stress injury in H9c2 cardiomyocytes

Objective:Yiqi Huoxue Decoction (YQHX) has been widely used for clinical treatment of ischemic heart disease.While oxidative stress plays a key role in the pathogenesis of ischemic heart disease,the function and molecular mechanism underlying antioxidative protective effects of YQHX on H9c2 cardiomyocytes against ischemia/hypoxia (I/H) have yet to be well clarified.Methods:H9c2 cells were subjected to 12 h of hypoxia with serum-free conditions and then treated with or without YQHX (100-400 μg/mL).Cell viability was examined using a CCK-8 assay.Maleic dialdehyde (MDA) and superoxide dismutase (SOD) activity were detected using commercial kits.Intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential were measured using fluorescence microscopy and confocal laser-scanning microscopy,respectively.Ultrastructural details of mitochondria in H9c2 cells were observed using transmission electron microscopy.The antioxidative protective pathway was assessed by measuring mRNA and protein expression of Nrf2 and HO-1,as well as AMPK activation.Results:I/H injury gradually induced oxidative stress.Treatment with YQHX significantly increased cell viability and reversed I/H-induced oxidative stress,including reducing the production of oxidative stress products (ROS and MDA),increasing SOD levels,improving mitochondrial morphology,and increasing mitochondrial membrane potential.YQHX was also observed to increase I/H-induced expression of Nrf2 and HO-1,and the activation effects of YQHX were blocked by an AMPK inhibitor.In addition,HPLC analysis showed that YQHX contained two active antioxidative constituents (calycosin and ferulic acid).Conclusion:The results suggest that anti-oxidative effects exerted by YQHX in H9c2 cardiomyocytes may be linked to upregulation of the AMPK-mediated Nrf2/HO-1 pathway.